Various procedures are offered for isolation of complex glycosphingolipids, but all the methods are based on the same principle of extraction and vary only with regard to solvent mixtures. The serious flaws of these procedures lie in limited potential of the solvents to penetrate and brake hydrophobic bonds holding glycosphingolipids in the tissue network, to remove these highly polar substances from aqueous phase and from the solvent denatured residue, where the glycosphingolipids are physically trapped. The goals of the proposed research are: to develop new procedure for glycosphingolipids isolation, to characterize the complex and tightly bound glycosphingolipids of the cell membranes, and to determine which glycosphingolipids are involved in the formation of protein-lipid complexes; to isolate cell organelles and determine the glycosphingolipids composition therein, to show subcellular location of an enzyme(s) involved in sulfation of complex glycosphingolipids, and in overall to broaden our knowledge of glycosphingolipids and their functions in the cell membrane. Gastric mucosa from hog and dog will be used to develop new isolation procedure and to study the chemistry of glycosphingolipids and their complexes with other cell membrane components. Subcellular fractionation will be performed to determine the glycosphingolipids composition of subcelluar fractions and to establish the location of the enzymes involved in the sulfation of complex glycosphingolipids.